Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes Shuhei Shima 1,2 , Hirokazu Matsui 2 , Satoshi Tahara 2 and Ryozo Imai 1 1 Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, NARO, Toyohira-ku, Sapporo, Japan 2 Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan Trehalose is a nonreducing disaccharide in which two glucose units are linked by an a,a -1,1-glycosidic link- age. The prevalent pathway for trehalose synthesis includes two enzymatic reactions. Trehalose 6-phos- phate (Tre6P) is generated from UDP-glucose and glu- cose 6-phosphate (Glc6P) in a reaction catalyzed by trehalose-6-phosphate synthase (TPS). Tre6P is then dephosphorylated to form trehalose via trehalose-6- phosphate phosphatase (TPP) [1]. In yeast, trehalose synthesis is carried out by a large enzyme complex that is composed of four subunits, including TPS1, TPS2, and regulatory subunits TSL1 and TPS3 [2]. Trehalose is widely distributed in nature. In bacteria, fungi, and insects, trehalose functions as a storage car- bohydrate or a blood sugar. In addition, trehalose can protect cellular integrity against a variety of environ- mental stresses associated with desiccation, heat, and cold [3]. In plants, the presence of trehalose has been Keywords functional analysis; kinetic analysis; Oryza sativa; recombinant protein; trehalose Correspondence R. Imai, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization, Hitsujigaoka 1, Toyohira-ku, Sapporo 0628555, Japan Fax ⁄ Tel: +81 11 857 9382 E-mail: rzi@affrc.go.jp (Received 8 November 2006, revised 14 December 2006, accepted 19 December 2006) doi:10.1111/j.1742-4658.2007.05658.x Substantial levels of trehalose accumulate in bacteria, fungi, and inverte- brates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a func- tional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzy- matic characterization of recombinant OsTPP1 and OsTPP2 revealed strin- gent substrate specificity for trehalose 6-phosphate and about 10 times lower K m values for trehalose 6-phosphate as compared with trehalose- 6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 °C for 4 min. These characteristics of rice trehalose-6- phosphate phosphatase enzymes are consistent with very low cellular sub- strate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses. Abbreviations ABA, abcisic acid; Glc1P, glucose 1-phosphate; Glc6P, glucose 6-phosphate; GST, glutathione S-transferase; TPP, trehalose-6-phosphate phosphatase; TPS, trehalose-6-phosphate synthase; Tre6P, trehalose 6-phosphate. 1192 FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS documented in a limited number of species, including Myrothamnus flabellifolia, a desiccation-tolerant desert plant [4,5], and Selaginella lepidophylla, a desiccation- tolerant moss [6]; its occurrence in many other plant species is uncertain. TPS and TPP genes were functionally identified in Arabidopsis thaliana by complementation of Saccharo- myces cerevisiae mutants [7,8]. Homologous TPS and TPP genes have now been identified in many other plant species. These results suggest that trehalose synthesis may in fact be ubiquitous among angio- sperms, although the levels to which it accumulates are generally low [1,9]. Attempts to increase trehalose content in plants by overexpressing microbial TPS and TPP genes resulted in transgenic tobacco and potato plants with increased stress tolerance at the tissue level [10–12]. However, these transformants exhibited pleiotropic phenotypes, such as stunted growth and lancet-shaped leaves [11,12]. On the other hand, expression of an Escherichia coli TPS– TPP fusion enzyme in transgenic rice resulted in accumulation of 3–10 times more trehalose compared to nontransgenic rice plants, imparting abiotic stress tolerance without altering morphology [13,14]. There- fore, these findings suggested that accumulation of Tre6P may result in the observed morphologic alter- ations in the tobacco and potato studies. Although trehalose biosynthesis in higher plants has been demonstrated, details of both the physiologic functions and regulation of this pathway remain lar- gely unknown. Genome sequencing of Arabidopsis and rice has revealed complex genomic organization of plant trehalose biosynthesis genes. Eleven putative TPS and 10 putative TPP genes were identified within the Arabidopsis genome, and nine putative TPS and nine putative TPP genes were found within the rice genome. Genetic studies have revealed that trehalose biosyn- thesis genes function specifically in regulating plant growth and development. An Arabidopsis knockout mutant of AtTPS1 exhibited impaired embryo matur- ation [15]. Further characterization of the mutant dem- onstrated that AtTPS1 is also required for vegetative growth and flowering [16]. A recent study established that a maize TPP gene is involved in inflorescence development [17]. A more specific function of trehalose biosynthesis in the regulation of starch biosynthesis has recently been revealed. Trehalose feeding was found to induce expression of ApL3, encoding a large subunit of ADP-glucose pyrophosphorylase in Arabid- opsis [18,19]. It was demonstrated recently that Tre6P directly regulated starch synthesis via post-transla- tional redox activation of ADP-glucose pyrophospho- rylase [20,21]. In our previous study, we demonstrated that expres- sion of the rice TPP gene OsTPP1 is rapidly and tran- siently induced by chilling stress and abcisic acid (ABA) treatment. Induction of OsTPP1 was followed by transient increases in total TPP activity and treha- lose content in rice root [22]. Eight other members of the rice TPP gene family have not yet been character- ized, so it is not known if these members have diver- gent functions in rice. In addition, the enzymatic properties of plant TPPs are largely unknown. In this article, we report the isolation of a second TPP gene from rice, OsTPP2, and its relative tran- scription in response to abiotic stresses, as well as the in vivo and in vitro functionality of its translated prod- uct. We also describe unique kinetic and biophysical properties of the plant TPPs. Results Isolation of rice OsTPP2 Completion of the rice genome sequence revealed nine putative TPP genes. To determine which of these TPP genes are expressed in rice seedlings, RT-PCR was car- ried out using specific primer sets designed to amplify transcripts from all of these OsTPP genes (OsTPP1– OsTPP9) [22]. Only mRNA for OsTPP2 was detected in addition to that of the previously characterized OsTPP1 after 28 cycles of PCR amplification (Fig. 1A). OsTPP3–OsTPP9 mRNAs were not detec- ted in root and shoot tissues after up to 35 cycles of amplification (data not shown). These results suggested that OsTPP1 and OsTPP2 were the major TPP genes expressed in rice seedlings. A full-length OsTPP2 cDNA was then isolated from root tissue by RT-PCR. The OsTPP2 gene contained an ORF encoding a 42.6 kDa protein with 382 amino acid residues. Overall amino acid sequence homology between OsTPP2 and OsTPP1 was 53% (Fig. 1B). Greater similarity was observed between OsTPP2 and Arabidopsis AtTPPA (57%). OsTPP2 contains two motifs shared by all TPP enzymes (Fig. 1B), known as phosphatase boxes: (FIL - MAVT)-D-(ILFRMVY)-D-(GSNDE)-(TV)-(ILVAM)- (ATSVILMC)-X-(YFWHKR)-X-(YFWHNQ) (domain A), and (KRHNQ)-G-D-(FYWHILVMC)-(QNH)- (FWYGP)-D-(PSNQYW) (domain B) [23]. Responses of OsTPP2 to chilling and other abiotic stresses In our previous study, we demonstrated that OsTPP1 expression is transiently induced by multiple abiotic stresses [22]. We therefore determined whether S. Shima et al. Rice trehalose-6-phosphate phosphatases FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS 1193 OsTPP2 expression is also responsive to abiotic stres- ses. RNA gel blot analysis was performed on total RNA extracted from rice seedlings subjected to low temperature (12 °C), drought, and salt stresses (Fig. 2). OsTPP2 mRNA levels were detectable prior to stress treatments, and transiently increased in response to low temperature, peaking at 10 h after the initiation of treatment in both shoot and root tis- sues. This expression pattern contrasted with the observed rapid induction of OsTPP1 and gradual induction of OsMEK1 in response to low-temperature treatment [22,24]. Drought stress transiently induced OsTPP2 expression, which peaked at 6 h in shoots and 2 h in roots (Fig. 2). The induction of OsTPP2 expression occurs earlier during stress treatment com- pared with expression of another drought-induced gene (salT) [25]. Treatment with 150 mm NaCl also induced OsTPP2 expression (Fig. 2) in roots, suggest- ing that stresses associated with water deficit similarly affect expression of this gene. However, in contrast to chilling and drought stress treatments, clear induction of OsTPP2 was not observed in shoots, whereas the salt treatment effectively induced salT in both roots and shoots. Slight and transient induction of OsTPP2 was observed in roots and shoots in response to exo- genous ABA. Together, these expression analyses indi- cated involvement of OsTPP2 in multiple stress responses. A B A B Fig. 1. Expression of putative OsTPP genes in young vegetative tissues, and alignment of TPP sequences. (A) Expression analysis of puta- tive TPP genes with RT-PCR, using RNAs extracted from root and shoot tissues of rice seedlings (O. sativa L. cv. Yukihikari). (B) Alignment of the amino acid sequences of OsTPP2, OsTPP1 (O. sativa [22]), AtTPPA and AtTPPB (A. thaliana [8]), TPS2 (Sa. cerevisae [34]) and OtsB (E. coli [35]). Database accession numbers are: OsTPP1, BAD12596; OsTPP2, BAF34519; AtTPPA, AAC39369; AtTPPB, AAC39370; TPS2, CAA98893; and OtsB, CAA48912. Shading reflects the degree of amino acid conservation. Black shading indicates amino acid identity. The bars represent highly conserved domains. Rice trehalose-6-phosphate phosphatases S. Shima et al. 1194 FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS OsTPP2 complements a yeast Dtps2 mutant To detect OsTPP2 enzyme function in vivo,aSa. cere- visiae (YPH499) tps2 (TPP) deletion mutant [22] was transformed with plasmid constructs based on the pAUR123 vector (Takara). Whereas wild-type cells grow at both 30 °C and 36 °C, growth of the Dtps2 mutant at 36 °C was inhibited because of its inability to synthesize trehalose (Fig. 3). The same mutant yeast strain transformed with OsTPP2 recovered wild-type levels of growth at 36 °C, suggesting that OsTPP2 is a functional TPP enzyme in yeast cells. TPP activity of recombinant OsTPP2 To determine whether OsTPP2 exhibits TPP activity in vitro, it was purified as a recombinant protein. To accomplish this, the ORF of OsTPP2 was inserted into a pGEX-6P-3 vector to produce a glutathione S-trans- ferase (GST)–OsTPP2 fusion protein. After affinity column purifications and protease digestion, OsTPP2 proteins were purified to near homogeneity. The size of the purified recombinant enzyme was estimated to be 45 kDa on SDS ⁄ PAGE, in accordance with the size deduced from the nucleotide sequence (42.6 kDa) (Fig. 4A). TPP activity was then measured using this purified recombinant enzyme. Aliquots of purified enzyme were added to the reaction mixtures, and conversion of Tre6P into trehalose was detected as a measure of enzyme activity (Fig. 4B). Under these same conditions, purified GST or NaCl ⁄ P i solution without enzyme did not result in this conversion (Fig. 4B). We therefore concluded that OsTPP2 encodes a functional TPP enzyme. Enzymatic properties of OsTPP1 and OsTPP2 Although genes encoding plant TPPs have been identi- fied in several plant species, their enzymatic character- istics have not been explored. To further characterize the enzymatic properties of plant TPP enzymes, we also purified recombinant OsTPP1 using the same methods. Then, the kinetic parameters of these recom- binant OsTPP1 and OsTPP2 enzymes were determined. The K m values for Tre6P of OsTPP1 and OsTPP2 were determined to be 0.0921 and 0.186 mm, respect- ively, using Hanes–Woolf plots (Table 1). The k cat val- ues of OsTPP1 and OsTPP2 were 6.52 and 13.4 s )1 , respectively. Therefore, the k cat ⁄ K m values of OsTPP1 and OsTPP2 were approximately the same. These Fig. 3. Complementation of the heat-sensitive phenotype of a Sa. cerevisiae tps2 deletion mutant by introduction of OsTPP2.A YPH499 tps2 deletion mutant was transformed with the pAUR123 vector (Dtps2) and pAUR123-OsTPP2 (Dtps2 ⁄ OsTPP2). As a posit- ive control, YPH499 wild-type cells were transformed with the empty pAUR123 vector (wt). These transformants were grown overnight in YPD liquid medium supplemented with 0.5 lgÆmL )1 aureobasidin A. The cultures were then diluted 1–1000 times. Five microliters of each dilution was then spotted onto an YPD agar plate supplemented with 0.5 lgÆmL )1 aureobasidin A. These plates were incubated at 30 °Cor36°C for 2 days. A B C D Fig. 2. Expression of OsTPP2 in rice seedlings in response to abiotic stress and exogenous ABA treatment. Total RNAs were iso- lated from rice seedlings subjected to chilling stress (A), drought stress (B), 150 m M NaCl stress (C), and exogenous ABA (50 lM) solution (D). The RNA blots were hybridized with an OsTPP2 probe. The expression of OsTPP1 is shown for comparison of expression patterns, and those of OsMEK1 [24] and salT [25] are shown as positive controls for these treatments. Ethidium bromide-stained total RNA (10 lg) is presented as a loading control. S. Shima et al. Rice trehalose-6-phosphate phosphatases FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS 1195 results indicated that both enzymes exhibit similar cat- alytic activities. It is interesting to note here that the K m values for rice TPPs are more than 10 times lower than those of bacterial TPP enzymes reported thus far. For instance, others reported that the K m values for E. coli and Mycobacterium smegmatis TPPs were 2.5 mm and 1.5 mm, respectively [26,27]. To determine the substrate specificity of these recombinant proteins, phosphatase activities were measured using various sugar phosphate substrates [glucose 1-phosphate (Glc1P), Glc6P, galactose 6- phosphate, mannose 1-phosphate, mannose 6-phos- phate, fructose 1-phosphate, fructose 6-phosphate, sucrose 6-phosphate, lactose 1-phosphate, and ribose 5-phosphate]. Both OsTPP1 and OsTPP2 exhibited strong phosphatase activity upon Tre6P, but almost no activity (less than 1% relative to Tre6P) was detec- ted with any of the other sugar phosphates tested (data not shown). The pH dependences of OsTPP1 and OsTPP2 enzyme activities were determined within a pH range of 5.5–9.0, using two different buffers (Mes ⁄ NaOH, pH 5.5–7.5; Tris ⁄ HCl, pH 7.0–9.0). The pH optima of OsTPP1 and OsTPP2 were approximately 7.0 and 6.5, respectively, whereas the enzymes had almost no activ- ity at pH 5.5 or 9 (Fig. 5). The heat stabilities of the recombinant OsTPP1 and OsTPP2 were determined by measuring residual activities after heat treatments (40–80 °C) (Fig. 6). A B Fig. 4. Purification of recombinant OsTPP1 and OsTPP2 and deter- mination of their activities. Recombinant OsTPP1 and OsTPP2 were purified according to the experimental procedure described previ- ously. (A) SDS ⁄ PAGE (12%) was run with protein standards (lane M), crude extracts of the recombinant bacterial strains induced without (lane 2) or with (lane 3) isopropyl thio-b- D-galacto- side, and purified recombinant OsTPP1 or OsTPP2 (lane 4). (B) Chromatograms detailing TPP activities of recombinant OsTPP1, OsTPP2, and GST. These proteins (0.5 lg) were used for assays in 100 lL reaction mixtures (2 m M Tre6P,2mM MgCl 2 ,50mM Tris ⁄ HCl, pH 7.0). T; trehalose; T6P, trehalose 6-phosphatase. Table 1. Enzymatic properties of recombinant OsTPP1 and OsTPP2. Protein K m a (mM) K cat (s )1 ) K cat ⁄ K m (mM )1 Æs )1 ) Reference OsTPP1 0.0921 6.52 70.8 This study OsTPP2 0.186 13.4 72.0 This study E. coli TPP 2.5 14.3 5.8 [27] M. smegmatis TPP 1.5 – – [26] a K m for Tre6P. Rice trehalose-6-phosphate phosphatases S. Shima et al. 1196 FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS Heat treatment at 50 °C or higher for 4 min nearly eliminated both OsTPP1 and OsTPP2 activity, indica- ting that both enzymes are heat-labile. Discussion Identification and functional characterization of treha- lose biosynthesis genes have established trehalose biosynthesis in higher plants. However, low-level accu- mulation of trehalose in plants suggests a distinctive function of this substance compared with its role in other organisms. Organization of these trehalose bio- synthesis genes is also quite unique in higher plants. Only one or two copies of TPS and TPP genes exist in most bacteria, fungi, and insects, whereas these genes constitute a large gene family in higher plants. For example, in Arabidopsis, 11 TPS and 10 TPP genes have been identified from genomic information [28,29], and nine TPS and nine TPP homologs are found in the rice genome. Therefore, researchers have specula- ted that trehalose biosynthesis is tightly regulated dur- ing plant growth and development, and that each TPS and TPP gene is under specific regulation. In this study, we identified a novel TPP gene (OsTPP2) from rice, and demonstrated that OsTPP2 and the previ- ously identified OsTPP1 are predominantly expressed in young vegetative tissues of rice. According to the results of yeast complementation analysis and enzyme assays with recombinant protein, OsTPP2 encodes a functional TPP. Expression of OsTPP2 was regulated by multiple stress factors, such as chilling, drought, and salt stresses, as well as ABA treatment (Fig. 2). It is interesting that OsTPP1 is also regulated by the same stress factors but its induction kinetics are quite different in comparison to those of OsTPP2 [22]. OsTPP2 is transiently induced after 10 h of chilling stress (12 °C), whereas transient induction of OsTPP1 occurs much earlier ) within 2 h of the chilling stress [22]. Similarly, the patterns of OsT- PP2 induction in response to drought and salt stresses A B Fig. 5. Optimal pH for recombinant OsTPP1 (A) and OsTPP2 (B) activity. The reaction buffer systems tested were Mes ⁄ NaOH (pH 5.5–7.5) and Tris ⁄ HCl (pH 7.0–9.0). The enzyme assay was car- ried out as described in Experimental procedures. This activity was determined by measuring inorganic phosphate released from Tre6P. A B Fig. 6. Heat stability of recombinant OsTPP1 (A) and OsTPP2 (B). The purified recombinant OsTPP1 and OsTPP2 were incubated for various time periods at temperatures ranging from 40 °Cto80°C. The residual activity after treatment is expressed as a percentage of the original activity. S. Shima et al. Rice trehalose-6-phosphate phosphatases FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS 1197 differ from those of OsTPP1 under similar conditions. OsTPP2 is induced by exogenous ABA during 1–2 h of treatment, whereas ABA induction of OsTPP1 occurs more rapidly (within 1 h) and pronouncedly [22]. These data clearly show that OsTPP1 and OsTPP2 are under distinctive regulation. Our preliminary results with green fluorescent protein fusion proteins suggested that both OsTPP1 and OsTPP2 are cytosolic proteins. It was therefore suggested that trehalose biosynthesis is tightly regulated in response to multiple abiotic stress factors in rice, the process involving two differentially regulated TPP enzymes. Using recombinant enzymes, we conducted the first detailed functional characterization of plant TPPs. These rice TPPs displayed three distinct properties com- pared with the previously characterized microbial TPPs. First, the K m values for the recombinant OsTPP1 and OsTPP2 enzymes are lower than values published for the microbial enzymes. Others have reported that the Tre6P concentration in Arabidopsis is relatively very low (10.1 ± 1.3 lgÆg )1 fresh weight) [30]. Therefore, these low K m values for OsTPP1 and OsTPP2 correlate with low concentrations of this substrate in plant cells. Second, these rice TPPs were overall less stable than bacterial enzymes. For example, others reported that a TPP from Mycobacterium did not lose activity after heat treatment at 60 °C for 6 min [31]. In contrast, the results of this study indicate that OsTPP1 and OsTPP2 are completely inactivated after incubation at 50 °C for 3 or 4 min, so these enzymes are heat-labile. This further suggests that the turnover rates of OsTPP1 and OsTPP2 are relatively high. Relatively rapid turnover of these enzymes would better enable tight control of trehalose or Tre6P levels in rice. Third, the substrate specificity of OsTPP1 and OsTPP2 is higher than for the correspond- ing bacterial enzymes [31]. For instance, the mycobacte- rial TPP exhibited approximately 18% and 5% relative activities against Glc1P and Glc6P, when compared with Tre6P substrate. In contrast, no phosphatase activ- ity was detected when OsTPP1 and OsTPP2 were incubated with various sugar phosphate substrates, including Glc1P and Glc6P (data not shown). The function of trehalose in stress tolerance has been documented in several transgenic plants [13,32]. However, whether trehalose has a direct stress protec- tion function in wild-type plants (as in the case of microorganisms) or a regulatory function remains unclear. Rice transgenic plants expressing an otsA–otsB fusion gene exhibited improved stress tolerance; how- ever, trehalose in these plants did not reach high enough levels to function as an osmoprotectant [14,33]. Moreover, trehalose accumulated in wild-type rice plants at very low levels, and changed minimally and transiently in response to chilling stress [22]. In this study, we discovered that stress-induced OsTPP2 expression was transient and distinct from the expres- sion pattern of salT (which encodes a protein with a putative stress protection function) under those same conditions [25]. Moreover, others have shown recently that the trehalose biosynthesis pathway is intercon- nected with the glucose and ABA signaling pathways in Arabidopsis [30]. These current studies suggest that trehalose or Tre6P is involved in regulation of stress responses in higher plants. Although the trehalose biosynthesis pathways are conserved during evolution, a unique function of this substance in higher plants has yet to be elucidated. Rather than overproduction of trehalose as a stress protectant or as a storage carbohydrate, fine-tuned biosynthesis is required to produce the putative signa- ling molecules trehalose or Tre6P in higher plants. Our genetic and biochemical analyses presented here sup- port this hypothesis. Experimental procedures Plant materials, growth conditions and stress treatments Seeds of Japonica rice (Oryza sativa L. cv. Yukihikari) were surface-sterilized in 70% ethanol for 20 min, further steril- ized in 2.5% sodium hypochlorite solution for 25 min, and then washed several times with sterile water. These steril- ized seeds were then soaked in distilled water for 4 days and set for germination in the dark at 20 °C. Germinated seeds were uniformly distributed onto a plastic mesh grid that was supported by a plastic container filled with water up to the base of the mesh grid. Seeds were then grown under continuous illumination in a growth chamber at 25 °C. After growth for 7 days, the seedlings were subjected to various abiotic stress treatments. Chilling treatment was imposed by transferring mesh grids containing seedlings into containers filled with prechilled water at 12 °Cina growth chamber set at the respective temperature. Roots and shoots of the treated seedlings were collected after 0 (control), 1, 2, 4, 6, 10, 24 and 48 h of chilling treatment, immediately frozen in liquid nitrogen, and stored at )80 °C for further analysis. For NaCl and ABA treatments, 7-day- old rice seedlings were transferred, along with the mesh grid, and placed into solutions containing 150 mm NaCl and 50 lm ABA, respectively. For ABA treatments, shoots were also sprayed with ABA solution. Drought treatment was imposed by shifting the mesh grid with seedlings (immediately removing free water from roots by blotting on a paper towel) into a container without water. Roots and shoots of the treated seedlings were then collected and stored as mentioned previously. Rice trehalose-6-phosphate phosphatases S. Shima et al. 1198 FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS RT-PCR and cDNA cloning Expression analysis of all putative TPP genes was carried out by RT-PCR. Total RNA was extracted from root and shoot tissues of rice seedlings (O. sativa L. cv. Yukihikari) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA (1 lg) was reverse-transcribed using the GeneAmp Gold RNA PCR Reagent Kit (Applied Biosys- tems, Foster City, CA, USA) with an oligo-dT primer. The following PCR was carried out using gene-specific forward and reverse primers (listed in Table 2) according to the protocol supplied with the kit. GeneAmp 9700 (Applied Biosystems) was used for amplification with the following program: 28 or 35 cycles of 94 °C for 45 s, 53 °C for 45 min, and 72 °C for 1.5 min, with a final extension at 72 °C for 5 min. The amplified bands were cloned into a pGEM-T easy vector (Promega, Madison, WI, USA) and subsequently sequenced. DNA sequencing and analysis DNA sequencing was carried out using an ABI PRISM 310 Genetic Analyzer (PE Biosystems, Foster City, CA, USA). The BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) was used for the sequencing reaction. Sequence analysis was performed using genetyx software (Software Development, Tokyo, Japan). Multiple amino acid alignments were performed using the online clustal w alignment program at a website maintained by DDBJ (http://www.ddbj.nig.ac.jp/search/clustalw-e.html). Northern blot analyses Total RNA was isolated from plant tissues using TRIzol reagent (Invitrogen). Ten micrograms of total RNA was then denatured in formamide and formaldehyde, separated on 0.8% agarose gels, and transferred onto Hybond-N + membranes (GE Healthcare, Piscataway, NJ, USA). The blots were hybridized in Rapid-Hyb Buffer (GE Health- care) at 65 ° C with a 32 P-labeled full-length OsTPP2 frag- ment as a probe. The blots were washed twice with wash buffer (2 · NaCl ⁄ Cit, 0.1% SDS) for 15 min at 65 °C, and then washed twice with another wash buffer (0.2 · NaCl ⁄ - Cit, 0.1% SDS) for 15 min at 65 °C. The blots were then exposed to X-ray film for signal detection. Yeast complementation A Sa. cerevisiae tps2 deletion mutant of the YPH499 (MATa his3-D200 leu2-D1 lys2-801 trp1-D1 ade2-101 ura3-52) strain was used as a host cell population for complementation analysis [22]. The OsTPP2 ORF region was cloned into the pAUR123 vector (Takara, Kyoto, Japan), which allows constitutive expression of the insert under control of the ADH1 promoter. Transformation of Sa. cerevisiae was carried out with the S.c. EasyComp Transformation Kit (Invitrogen). The plasmid vectors pAUR123-OsTPP2 and pAUR123 were transformed into both the wild-type and the mutant YPH499 strains. These transformants were cultured in YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose) until a D 600 of 0.5 was reached. The collected cells were resuspended in sterilized water, and a series of dilutions (10 )1 ,10 )2 , and 10 )3 ) was made. Five microliters of each dilution was then dropped onto YPD plates and cultured for 2 days at either 30 °Cor36°C. Recombinant protein production and purification The ORFs of OsTPP1 and OsTPP2 were PCR-amplified with BamHI and SalI linker sequences from the correspond- ing cDNA clones. These PCR products were digested and ligated with a predigested pGEX-6P-3 vector (GE Health- care). E. coli BL21 cells were then transformed with the resulting pGEX-OsTPP1 and pGEX-OsTPP2 vectors, respectively. The transformant cells were grown overnight in LB medium containing ampicillin (50 lgÆmL )1 ), inoculated into 2 · YT medium containing ampicillin (50 lgÆmL )1 ), and cultured at 37 °C for 3 h. Recombinant protein expres- sion was induced with 0.5 mm isopropyl thio-b-d-galacto- side and incubated for another 3 h. Pelleted cells were resuspended in 10 mL of NaCl ⁄ P i (pH 7.4, 0.14 m NaCl, 3mm KCl, 10 mm Na 2 HPO 4 ,2mm KH 2 PO 4 ) and disrup- ted with sonication. Lysed samples were centrifuged in an AR 0/5-24 rotor (MX-300; Tomy, Tokyo, Japan) at 14 000 g for 5 min at 4 °C. Recombinant proteins were purified from the soluble fractions with a glutathione-seph- arose 4B affinity column (GE Healthcare), and then digested with precision protease at 4 °C. The protein samples were separated by SDS ⁄ PAGE (12%) and stained with Coomas- Table 2. Oligonucleotide primers used for RT-PCR analysis. Gene Orientation Oligonucleotide sequence (5¢-to3¢) OsTPP1 (AB120515) Forward TCAGTCATGCCCGGTGGC Reverse ACACTGAGTGCTTCTTCC OsTPP2 (AB277360) Forward ATGGATTTGAAGACAAGCAAC Reverse TTAAGTGGATTCCTCCTTCCA OsTPP3 (AP004341) Forward ATGACGAACCACGCCGGC Reverse CTACTTGCCAATCAGCCCTTT OsTPP4 (AP004119) Forward CTGTTCGTCTCGACGAGT Reverse TCTTACGGCCTCTACACC OsTPP5 (AL606633) Forward CACGCACCTACACCAAGA Reverse TGATGGGCCTCTCAGCAT OsTPP6 (AP004658) Forward TCAACGGATGGGTGGAGT Reverse ACTTGGACACGAGGATGC OsTPP7 (AP005580) Forward CACGACGCTGTTCCCGTA Reverse TCAACCGTGTCCTGGACA OsTPP8 (AP004727) Forward AGTACGACGCGTGGACGA Reverse GTGTGCTGCGAAGTCATG OsTPP9 (AC103551) Forward TGCTCTCTCGCTCTCGTT Reverse AGTGTCACTGTGGTCAGG S. Shima et al. Rice trehalose-6-phosphate phosphatases FEBS Journal 274 (2007) 1192–1201 ª 2007 The Authors Journal compilation ª 2007 FEBS 1199 sie Brilliant Blue. The protein concentrations were measured with a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using IgG as a standard. Assay of TPP activity Assays for TPP enzyme activity were carried out in reaction mixtures (100 lL) containing the following components: 2mm Tre6P,2mm MgCl 2 ,50mm Tris ⁄ HCl buffer (pH 7.0), and an appropriate amount of enzyme (0.5 lg). After incubation at 37 °C for 30 min, these mixtures were boiled for 4 min to stop the reaction. The amount of treha- lose produced was determined using a Dionex (DX-500) gradient chromatography system coupled with pulse amper- ometric detection (Dionex Corporation, Sunnyvale, CA, USA). The samples were applied to a CarboPac PA1 colum (Dionex) equilibrated with 0.1 m NaOH, using a flow rate of 1 mLÆmin )1 . A 0.2 m sodium acetate gradient buffered in 0.1 m NaOH was applied over 3–8 min; the sodium acetate concentration was then increased to 1 m for 2 min before equilibrating the column again with 0.1 m NaOH. Under these conditions, the retention times of trehalose and Tre6P were 2.8 min and 12.0 min, respectively. For the analysis of optimum pH, substrate specificity, and heat tolerance, TPP activity was assayed by determin- ing released inorganic phosphate levels with BIOMOL GREEN Reagent (Biomol Research Laboratories, Plymouth Meeting, PA, USA). Two volumes of this reagent were added to each terminated enzyme reaction, and then incubated for 20 min at room temperature. The absorbance of each mixture was determined at 620 nm with a Beckman DU-65 spectrophotometer (Beckman Instrument, Inc., Full- erton, CA, USA) and compared with that of a standard solution. Substrate specificity TPP enzymes (0.5 lg) were added to a reaction mixture (100 lL) containing 50 mm Tris ⁄ HCl (pH 7.0), 2 mm MgCl 2 , and 2 mm sugar phosphate, and incubated at 37 °C for 30 min. The sugar phosphate substrates tested were Glc1P, Glc6P, galactose 6-phosphate, mannose 1-phos- phate, mannose 6-phosphate, fructose 1-phosphate, fructose 6-phosphate, sucrose 6-phosphate, lactose 1-phosphate and ribose 5-phosphate. All sugar phosphates were purchased from Sigma Chemical Co. (St Louis, MO, USA). PH optimum The pH optimum of TPP was determined using two differ- ent buffer systems, Mes ⁄ NaOH (pH 5.5–7.5) and Tris ⁄ HCl (pH 7.0–9.0). The conditions for the enzyme reaction and determination of inorganic phosphate levels were as des- cribed above. Heat stability of TPP The purified proteins were heat-treated at different temper- atures (40–80 °C) for 0, 1, 2, 3 or 4 min, and then cooled immediately on ice. After centrifugation at 20 000 g for 5 min by using MX-300 (Tomy), the supernatants were used to determine residual activity. Enzyme reactions and determination of inorganic phosphate levels were carried out as described above. References 1 Goddijn OJ & van Dun K (1999) Trehalose metabolism in plants. 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